Group of nanobioengineering

Bioengineering department

Head: Oksana Nekrasova, Ph.D.
okatja@yandex.ru+7(495)3306638

bioengineering; Membrane proteins; Ligands; Ligand-receptor interactions

The group is aimed at the bioengineering of recombinant proteins and polypeptides, developing the procedures for their purification and renaturation, studies on the properties of recombinant molecules using physico-chemical methods, as well as the approaches and methods of nanotechnology.

The group collaborates with the departments of the IBCh RAS (Laboratory of optical microscopy and spectroscopy of biomolecules, the Molecular Instruments for Neurobiology Group and the Laboratory of molecular toxinology) and with Department of Bioengineering of the Biological Faculty of the Lomonosov Moscow State University and the Laboratory of Physicochemical Foundations of Reception of the Emanuel IBHF RAS.

Bioengineering group was formed in 2010 as a part of Bioengineering department of IBCh RAS.

  • Membrane engineering in living cells – bioengineering of recombinant receptor molecules that are targeted to and inserted into bacterial membrane, study of their ligand-binding activity at the surface of the cell membrane using fluorescent methods of detection. 
  • Development of bioengineering approaches to the production of recombinant membrane photosensitive proteins in order to study their structural organization and photochemical properties.
  • Development of bioengineering methods for obtaining soluble protein and peptide ligands in the functionally active form and studying their interaction with receptor proteins.

1.     A bioengineering test system was developed to study interactions of potassium channels with ligands. The test system is based on the use of fluorescently-labeled peptide probe and hybrid potassium channels that are embedded in the membrane of whole bacterial cells. The detection of ligand-receptor interactions is carried out by the method of laser scanning confocal microscopy (LSCM). Using the test system new blockers originating from animal venoms were identified that target Kv1.1 and Kv1.3 – potassium channels of important biomedical significance. With the help of molecular modeling methods, molecular determinants of interaction of peptide toxins with potassium channels were studied, and the mutant forms of peptide toxins were constructed with increased selectivity for the target channel. The principle of engineering of genetically encoded fluorescent ligands of potassium channels was developed with the aim to use them as fluorescent probes in binding studies and also for visualization of potassium channels in cells and tissues. The work is carried out in collaboration with departments of  IBCh - Laboratory of Optical Microscopy and Spectroscopy of Biomolecules, Laboratory of Molecular Instruments for Neurobiology, Laboratory of Molecular Toxinology, as well as Department of Bioengineering of the Biological Faculty of the Lomonosov Moscow State University.

2.     Bioengineering method was worked out for over-production of recombinant bacteriorhodopsin (Halobacterium salinarum) in E.coli expression system. Photochemical properties of recombinant bacteriorhodopsin were found to be similar to those of the monomeric form of the native protein. Recombinant bacteriorhodopsin and its mutants are used to study primary events of photocycle and mechanisms of energy transfer using the method of Femtosecond Transient Absorption Spectroscopy (in collaboration with the Laboratory of Physico-Chemical Basis of Reception, N.M. Emanuel Institute of Biochemical Physics RAS).

3.     New effective bioengineering methods have been developed for producing functionally active recombinant ligands, such as: disulfide-rich peptide toxins from scorpion venoms; efrin A1 - the ligand of  Ephrine receptors. The obtained peptides and proteins are used in various studies on ligand-receptor interactions. 

NamePositionContacts
Oksana Nekrasova, Ph.D.headokatja@yandex.ru+7(495)3306638
Elena Krjukovar. f.kelen.kryukova@yandex.ru+7(495)330-69-83
Kseniya Kudryashova, Ph.D.r. f.rekamoskva@mail.ru
Klara Birikh, Ph.D.sen. eng.+7(495)336-42-00
Sergej Yakimovres. eng.+7(495)330-72-74

Former members:

Roman Tikhonov, Ph.D.s. r. f.

All publications (show selected)

Loading...

Oksana Nekrasova

  • Russia, Moscow, Ul. Miklukho-Maklaya 16/10 — On the map
  • IBCh RAS, build. 52, office. 264
  • Phone: +7(495)3306638
  • E-mail: okatja@yandex.ru

Mechanisms of anticancer action of curaxins

In collaboration with Laboratory of optical microscopy and spectroscopy of biomolecules

Data of spFRET analysis support  the hypothesis that  anticancer drug curaxin, namely, its  CBL0137 derivative, can  affect long-distance enhancer-promoter communication (EPC) in chromatin by disrupting nucleosome structure or affecting the structure and dynamics of the linker DNA supporting efficient EPC (Kantidze et al., Nat Commun., 2019,10(1):1441). The data indicate also that CBL0137 attracts human FACT (protein factor that FAcilitates Chromatin Transcription) to nucleosomes, mediates hFACT-induced scaled, partially reversible nucleosome unfolding (or uncoiling of the nucleosomal DNA) and traps hFACT on nucleosomes. This curaxin-dependent FACT trapping can be a reason of hFACT redistribution from the transcribed chromatin regions to other genomic loci and contribute to the anticancer action of curaxins (Chang et al. Science Advances, 2018, 4 (11), eaav2131).

The studies were performed jointly with the specialists from the Institute of Gene Biology RAS (Kantidze O.L., Luzhin A.V., Golov A.K., Velichko A.K.),   Biology Faculty of Lomonosov Moscow State University (Valieva M.E., Lyubitelev A.V., Razin S.V.), Fox Chase Cancer Center, USA (Nizovtseva E.V., Studitsky V.M., Kulaeva O.I., Chang H.-W.), Roswell Park Comprehensive Cancer Center, USA (Gurova K.V., Safina A., Wang J.,), Eunice Kennedy Shriver National Institute for Child Health and Human Development, USA (Chereji R.V.), Rutgers University, USA (Morozov A.V.).

Publications

  1. Kantidze OL, Luzhin AV, Nizovtseva EV, Safina A, Valieva ME, Golov AK, Velichko AK, Lyubitelev AV, Feofanov AV, Gurova KV, Studitsky VM, Razin SV (2019). The anti-cancer drugs curaxins target spatial genome organization. Nat Commun 10 (1), 1441
  2. Chang HW, Valieva ME, Safina A, Chereji RV, Wang J, Kulaeva OI, Morozov AV, Kirpichnikov MP, Feofanov AV, Gurova KV, Studitsky VM (2018). Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins. Sci Adv 4 (11), eaav2131

MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity

In collaboration with Laboratory of optical microscopy and spectroscopy of biomolecules,  Group of in silico analysis of membrane proteins structure,  Laboratory of Molecular Instruments for Neurobiology

Оksana V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling), A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

KV1.2 Сhannel-Specific Blocker from Scorpion Venom: Structural Basis of Selectivity

In collaboration with Laboratory of optical microscopy and spectroscopy of biomolecules,  Laboratory of Molecular Instruments for Neurobiology,  Group of in silico analysis of membrane proteins structure

Laboratory of Molecular Instruments for Neurobiology is known for systematic study of Arthropods’ venoms and derived peptides that specifically target various ion channels. Scorpions’ venom is abundant with potassium channels (Kv) blockers, and this diversity was described in previously released in Kalium database.

In cooperation with Laboratory of optical microscopy and spectroscopy of biomolecules and Group of nanobioengineering an unique screening system permitted identification in the Mesobuthus eupeus scorpion venom of Kv1.2 blocker: peptide MeKTx11-1 binging with high affinity (IC50 ≈0,2 nM) and specificity (effect on Kv1.1, 1.3 and 1.6 emerges at >100-fold higher concentrations). This peptide differs from the related MeKTx11-3 by just two residues, possessing substantially lower Kv1.2-specificity.

Finally, Group of in silico analysis of membrane proteins structure conducted a molecular modeling study of these two peptides interacting with Kv1.2 channel, immersed into an explicit lipid bilayer. This study uncovered mechanism of selective action of MeKTx11-1 peptide. The developed analysis technique will be of use for future design of selective ligands of Kv and other channels, which may be applied in fundamental studies of molecular basis of nervous system function and as drugs prototypes.

MeKTx11-1, Kv1.2 channel –specific peptide blocker from the M.eupeus scorpion venom: structural basis of selectivity

In collaboration with Laboratory of biomolecular modeling,  Group of in silico analysis of membrane proteins structure,  Laboratory of Molecular Instruments for Neurobiology,  Laboratory of optical microscopy and spectroscopy of biomolecules

A.V. Feofanov (Laboratory of optical microscopy and spectroscopy of biomolecules), О.V. Nekrasova, K.S.Kudryashova (Group of nanobioengineering, Bioengineering department), A.A. Vassilevski, A.I. Kuzmenkov, A.M. Gigolaev (Laboratory of molecular instruments for neurobiology), A.O. Chugunov, V.M. Tabakmakher, R.G. Efremov (Group of in silico analysis of membrane proteins structure, Laboratory of biomolecular modeling).

A unique high-affinity and highly selective peptide blocker of Kv1.2 channel, MeKTx11-1, from the scorpion venom Mesobuthus eupeus was studied. Peptide MeKTx11-1 and its mutant forms were produced in a recombinant form, and their receptor-binding activity was studied against a panel of Kv1-channels. Molecular modeling of interaction of these peptides with Kv1.2 channel was carried out, and key structural elements of the interactions were determined. Peptide MeKTx11-1 may be used as a novel efficient molecular tool in neurobiology to identify and study the activity of Kv1.2 channel in the presence of different isoforms of Kv1-channels.

In collaboration with S.Peigneur and J.Tytgat fromUniversity of Leuven, Belgium and A.F. Fradkov from Evrogen JSC.

Scorpion venom is rich in peptide blockers of voltage-gated potassium channels (KV), and we have reflected this diversity previously in Kalium, a database dedicated to such peptides. A high-affinity and selective blocker of KV1.2 channels, characteristic of the human central nervous system, was obtained from the venom of the scorpion Mesobuthus eupeus. Using molecular modeling and site-directed mutagenesis, the mechanism of selective interaction between the toxin and channels was investigated.

An efficient method for production of recombinant α-КТх peptides – the blockers of potassium channels

In collaboration with Laboratory of optical microscopy and spectroscopy of biomolecules

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides: high yield of the target peptides (12-22 mg/l culture); retaining the native amino acid sequence of α-КТх peptides; high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds; simple and reliable procedure of peptide isolation and purification. The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical value.

AN EFFICIENT METHOD FOR PRODUCTION OF RECOMBINANT α-КТХ PEPTIDES – THE BLOCKERS OF POTASSIUM CHANNELS

In collaboration with Laboratory of optical microscopy and spectroscopy of biomolecules

O.V.Nekrasova, K.S.Kudryashova, S.A.Yakimov, M.P.Kirpichnikov

A.V.Feofanov

A bioengineering method for production of peptide blockers of potassium Kv1 channels has been developed that provides:

  • high yield of the target peptides (12-22 mg/l culture);
  • retaining the native amino acid sequence of α-КТх peptides;
  • high yield of the renatured form of the peptides with correctly formed three and four disulfide bonds;
  • simple and reliable procedure of peptide isolation and purification.

The recombinant peptides of the α-KTx family obtained by this method have the activity of the natural blockers. High affinity potassium channel blockers from scorpion venom are widely used to study the structure and function of the channels and have a promising medical significance.

Development of integrated transcriptomic and proteomic approach to search for blockers of potassium channels in animal venoms

Authors: 

Kuzmenkov A.I. , Vassilevski A.A., Grishin Eu.V.

Department of molecular neurobiology

Kudryashova K.S., Nekrasova O.V., Kirpichnikov M.P. 

Bioengineering Department  

Feofanov A.V. 

Laboratory of optical microscopy and spectroscopy of biomolecules

Annotation: 

An original approach was developed to search for new ligands of potassium channels. It combines the bioengineering cellular test system and transcriptomic and proteomic analysis of animal venoms. Using this approach eight high-affinity peptide blockers of voltage-gated potassium channel Kv1.1 (including five new peptides) were found in the venom of the scorpion Mesobuthus eupeus. The proposed approach is a versatile and effective tool for directed search for  blockers of potassium channels in natural venoms.